Transfected DrosophUa melanogaster cellscan express large quantities of class I major histocompatibility complex molecules. Such molecules lack endogenous peptides because the Drosophila cells are devoid ofproteins necessary for intracellular peptide loading. The empty molecules are efficiently expressed on the cell surface and can acquire extracellular peptides. The conformation and stability ofempty murine class I molecules are determined by the source of P2-microglobulin. All ,2-microglobulin-induced conformers of empty heavy chains seem to be unified in a common rigid conformation on peptide binding.

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We have isolated cytotoxic T lymphocytes (CTL) preferentially reactive with the al external domain of the H-2Ld antigen by selecting for T cells capable of recognizing a variant major histocompatibility complex (MHC) class I antigen sharing al sequences with H-2Ld. Using these CTL, we demonstrate that a synthetic al peptide corresponding to one of the helices derived from the H-2Ld molecule can be presented by a class I restriction element to reconstitute a CTL determinant borne by intact H-2Ld. Moreover, several other H-2L-reactive CTL generated independently were also able to recognize H-2Ld either as an intact alloantigen or as a peptide in coiijunction with appropriate class I restriction elements. These data demonstrate that an H-2 peptide can reconstitute a CTL target structure and suggest that some alloreactive T cells in fact might be directed against allogeneic class I peptides in the context of a class I framework.

We have examined mechanisms involved in gene transfer, protein expression, and antigen presentation after direct administration of retroviral vectors using a variety of antigen systems. We have identified transduced infiltrating cells at the injection site, and the majority of the infiltrating cells were of the monocyte͞macrophage lineage. We found that the splenic dendritic cell fraction contained proviral DNA, expressed antigenic proteins, and was able to present antigens efficiently to the immune system. Furthermore, the dendritic cell fractions from retroviral vectorimmunized mice were able to prime naive T cells in vitro, and adoptive transfer of in vitro-transduced dendritic cell fractions elicited antigen-specific cytotoxic T lymphocytes. These data suggest a role for dendritic cells in induction of immune responses elicited by retroviral vector-mediated gene transfer.We have developed Moloney murine leukemia virus-based retroviral vectors encoding therapeutic genes and have been studying the induction of immune responses mediated by these retroviral vectors. We have demonstrated that successful induction of immune responses could be achieved in mice (1), nonhuman primates (2), and humans (3), by injection of ex vivo vector-transduced autologous fibroblasts. These studies led us to investigate whether immune responses could be elicited by direct administration of retroviral vector by intramuscular injection. We have recently demonstrated induction of immune responses after direct administration of retroviral vectors intramuscularly in mice, rhesus monkeys, and baboons (4).Direct injection of retroviral vector offers several advantages over the ex vivo approach, primarily the elimination of the extensive efforts involved in generating autologous ex vivo fibroblast cell lines for each patient. With the ex vivo approach, the target antigen is exclusively expressed by transduced fibroblasts; however, direct administration of retroviral vectors can lead to the transduction of cells near the injection site as well as those cells residing in tissues to which vector can be transported. Consequently, it was not clear which cells were transduced by the retroviral vector, and which cells were responsible for presentation of retroviral vector-encoded antigens. In this paper, we have attempted to identify the vector-transduced cells and cells capable of presenting antigen to delineate mechanisms for induction of immune responses after direct injection of retroviral vectors.We have used retroviral vectors encoding HIV env͞rev, E. coli ␤-galactosidase (␤-gal), chicken ovalbumin, and firefly luciferase to identify the subsets of cells involved in antigen presentation and induction of immune responses. These four different antigen systems were used to verify that the mechanisms of in vivo transduction and induction of immune responses are comparable among retroviral vectors derived from the same packaging cell line and backbone construct regardless of the specific antigen they encode. Furthermore, each of these re...

We have analyzed changes in the antigenicity of major histocompatibility complex class I molecules resulting from the association of human beta-2 micro-globulin (B2m) with the mouse class I heavy chain. In particular, the H-2Ld molecule exhibited enhanced cross-reactivity for the 34-1-2 monoclonal antibody. In order to assess the nature of this structural alteration induced by human B2m, we utilized H-2 class I hybrid molecules in the mapping of the 34-1-2 determinant to the helical region of the alpha-1 domain. H-2Ld class I hybrid molecules were then used to establish the importance of the alpha-2 and -3 domains in the observed increase of 34-1-2 cross-reactivity following exchange with human B2m. The H-2Ld hybrids suggest that alterations in interdomain contact are responsible for enhanced 34-1-2 cross-reactivity on the H-2Ld molecule. It is likely that this alteration arises through changes in class I conformation at regions of the molecule distant from points of contact between B2m and the class I molecule. This suggests that perturbations induced by association of human B2m with H-2Ld can affect the conformation of the alpha-1 and -2 superdomain. That class I antigenic determinants are altered by the association of human B2m with mouse class I further suggests that the class I molecule is structurally flexible and may reflect the ability of the class I molecule to bind and present a vast array of disparate peptides to the T-cell receptor.